title: A strain-specific segment of the RNA-dependent RNA polymerase of grapevine fanleaf virus determines symptoms in Nicotiana species.
abstract: Factors involved in symptom expression of viruses from the genus Nepovirus in the family Secoviridae such as grapevine fanleaf virus (GFLV) are poorly characterized. To identify symptom determinants encoded by GFLV, infectious cDNA clones of RNA1 and RNA2 of strain GHu were developed and used alongside existing infectious cDNA clones of strain F13 in a reverse genetics approach. In vitro transcripts of homologous combinations of RNA1 and RNA2 induced systemic infection in Nicotiana benthamiana and Nicotiana clevelandii with identical phenotypes to WT virus strains, i.e. vein clearing and chlorotic spots on N. benthamiana and N. clevelandii for GHu, respectively, and lack of symptoms on both hosts for F13. The use of assorted transcripts mapped symptom determinants on RNA1 of GFLV strain GHu, in particular within the distal 408 nt of the RNA-dependent RNA polymerase (1E(Pol)), as shown by RNA1 transcripts for which coding regions or fragments derived thereof were swapped. Semi-quantitative analyses indicated no significant differences in virus titre between symptomatic and asymptomatic plants infected with various recombinants. Also, unlike the nepovirus tomato ringspot virus, no apparent proteolytic cleavage of GFLV protein 1E(Pol) was detected upon virus infection or transient expression in N. benthamiana. In addition, GFLV protein 1E(Pol) failed to suppress silencing of EGFP in transgenic N. benthamiana expressing EGFP or to enhance GFP expression in patch assays in WT N. benthamiana. Together, our results suggest the existence of strain-specific functional domains, including a symptom determinant module, on the RNA-dependent RNA polymerase of GFLV.
authors.affiliation: Université de Strasbourg, UMR 1131 'Santé de la Vigne et Qualité du Vin', 68021 Colmar, France.|INRA, UMR 1131 'Santé de la Vigne et Qualité du Vin', 68021 Colmar, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Vigne
authors.affiliation: Department of Plant Pathology and Plant-Microbe Biology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA.
authors.forename: John
authors.initials: J
authors.lastname: Gottula
authors.affiliation: Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg, France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Schmitt-Keichinger
authors.affiliation: Université de Strasbourg, UMR 1131 'Santé de la Vigne et Qualité du Vin', 68021 Colmar, France.|INRA, UMR 1131 'Santé de la Vigne et Qualité du Vin', 68021 Colmar, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Komar
authors.affiliation: Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg, France.
authors.forename: Léa
authors.initials: L
authors.lastname: Ackerer
authors.affiliation: Université de Strasbourg, UMR 1131 'Santé de la Vigne et Qualité du Vin', 68021 Colmar, France.|INRA, UMR 1131 'Santé de la Vigne et Qualité du Vin', 68021 Colmar, France.
authors.forename: Lorène
authors.initials: L
authors.lastname: Belval
authors.affiliation: Université de Strasbourg, UMR 1131 'Santé de la Vigne et Qualité du Vin', 68021 Colmar, France.|INRA, UMR 1131 'Santé de la Vigne et Qualité du Vin', 68021 Colmar, France.
authors.forename: Lalaina
authors.initials: L
authors.lastname: Rakotomalala
authors.affiliation: Université de Strasbourg, UMR 1131 'Santé de la Vigne et Qualité du Vin', 68021 Colmar, France.|INRA, UMR 1131 'Santé de la Vigne et Qualité du Vin', 68021 Colmar, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Lemaire
authors.affiliation: Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Ritzenthaler
authors.affiliation: Department of Plant Pathology and Plant-Microbe Biology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Fuchs
mesh_terms: D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D017860:Nepovirus / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity*; D010802:Phylogeny; D010935:Plant Diseases / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014026:Nicotiana / Q000821:virology*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D027843:Vitis / Q000821:virology*
chemical_list: D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase